Detection and amplification of nucleic acids play important roles in genetic analysis, molecular diagnostics, and drug discovery. Many such applications require specific, sensitive and cost effective quantitative detection of DNA mutations, copy number variants, gene expression or DNA methylation patterns that are present in a small fraction of total polynucleotides. In the field of cancer diagnosis for instance early detection of somatic mutations greatly increase the survival rate of cancer patients. Monitoring for occurrence of drug resistant mutations is also crucial in determining if a patient will have a relapse of the disease. An ideal example would be EGFR T790M mutation which occurs as a tyrosine kinase inhibitor resistant mutation in several non-small cell lung cancer (NSCLC) patients. Initially NSCLC patients harboring activating mutations in the epidermal growth factor receptor (EGFR) kinase domain tend to respond well to the tyrosine kinase inhibitors, gefitinib and erlotinib. However within a year in most cases relapse occurs due to drug resistance caused by an acquired secondary EGFR kinase domain mutation, T790M. Many of these early somatic mutations and drug resistant mutations are rare mutations which occur within a huge background of non-mutated DNA molecules. Many current methods use polymerase chain reaction (PCR), quantitative PCR (qPCR) and Next Generation Sequencing (NGS) to detect and quantify DNA and RNA variants from clinical samples.
While the performance of qPCR and NGS assays is constantly improving, the sensitivity and specificity of such methods suffer from technical limitations that make the methods inadequate for some applications, such as in detection and discrimination of rare DNA molecules with a single base mutation in situations when they are mixed with thousands of non-mutated DNA molecules. The sensitivity of existing qPCR and NGS technologies are typically limited to 1% and 5% respectively, which is not sufficient for rare allele detection. Another limitation with the current qPCR assays is the ability to combine multiple mutation detection assays into one multiplex diagnostic assay. Multiple mutation detection requires multiple primers and probes which can cause either non-specific amplification of DNA or lead to formation of primer-dimers which greatly reduces the efficiency of a qPCR assay.
There thus remains a need for the development of allelic enrichment and amplification strategies which will enable detection of rare mutations with high sensitivity and specificity. There also remains a need for development for single tube multiplex diagnostic assays which can greatly reduce the cost and time per assay.